Abstract

The Hh signaling pathway has been implicated in the formation and maintenance of many human cancers, including colon cancer. The canonical signaling pathway is initiated by ligation of the secretory Hh molecules to the transmembrane receptor, Patched, thereby releasing the inhibition of Smoothened (Smo), which then transduces the signal to a cytoplasmic complex causing the activation of Gli1 and 2. The active Gli molecules then translocate to the nucleus and function as transcription factors modulating Hh target gene expression. Non-canonical pathways such as Ras-MEK and PI3K-Akt can also activate Gli molecules as observed in lymphomas. Hh pathway inhibition with Cyclopamine, a natural inhibitor of Smo, has not been effective in clinical trials. We compared the efficacy of Cyclopamine to GANT61, a synthetic small molecule Gli antagonist, in inducing apoptosis in cc and investigated whether GANT61 modulated the effect of the cytotoxic agents currently used in colon cancer therapy. We studied the effects of Cyclopamine and GANT61 in the cc HT29, VRC5/c1 and HCT8 by measuring cell death. Our data demonstrate that the cc are not sensitive to Cyclopamine whereas treatment with GANT61 induces cell death in these cells by 48 hr post-treatment with an IC50 of 20-30 µM. The effect of GANT61 is independent of p53 function since two of the cc, HT29 and VRC5/c1, express mutant p53, while HCT8 has wild type p53. We have previously reported that interferon-gamma (IFN-gamma) potentiates the effects of 5-Fluorouracil/Leucovorin (FUra/LV), which are the cytotoxic agents currently used for treating colon cancer, and has a Fas-dependent component. Co-treatment of VRC5/c1 cells with FUra/LV/IFN-gamma and GANT61 (10 µM) significantly increased cell death in these cells 72 hr post-treatment. Co-treatment of HT29 with GANT61 (10 µM) and IFN-gamma (100 U/ml) induced cell death within 48 hr post-treatment, while treatment with the agents individually at the same concentrations had no cytotoxic effect. Furthermore, analysis of the Fas death receptor showed that co-treatment of HT29 cells with GANT61 and IFN-gamma at the same concentrations markedly increased the steady state level of Fas protein within 24 hr suggesting that the synergistic cytotoxicity is mediated via Fas signaling. These observations suggest that activated Gli activity causes repression of Fas receptor protein expression and that GANT61 functions synergistically with IFN-gamma to relieve the repression thereby inducing Fas signaling leading to apoptosis of cc. Collectively, these data suggest that Gli is activated in cc independent of Smo activation. Most importantly, careful combination of the Gli antagonist, GANT61 with currently used cytotoxic agents including FUra/LV and IFN-gamma may evolve into improved therapeutic strategies for colon cancer. Supported by NCI awards CA32613 and CA108929 to JAH.